POS0469 ENDOTHELIAL TO MESENCHYMAL TRANSITION AND SENESCENCE ARE PART OF THE FIBROTIC PATHOGENESIS IN SYSTEMIC SCLEROSIS
Authors: Y. H. Chiu, J. Spierings, J. M. Van Laar, J. De Vries-Bouwstra, M. Van Dijk, R. Goldschmeding
Journal: Annals of the Rheumatic Diseases (2022)

Background Systemic sclerosis (SSc) is a systemic autoimmune disease characterised by inflammation, vasculopathy and fibrosis. Several mechanisms, including endothelial to mesenchymal transition (EndMT) and cellular senescence, may be included in the fibrosis process, especially in response to inflammation and vasculopathy. Objectives Our study aimed to identify how EndMT and cellular senescence manifest in SSc skin fibrosis. Methods We performed a cross-sectional biobank study on formalin-fixed, paraffin-embedded skin biopsies from patients meeting the ACR/EULAR 2013 classification criteria for SSc. The extent of dermal fibrosis and inflammatory cell infiltration was scored on haematoxylin-eosin stained samples. Fibrosis severity was semi-quantified by the compactness of collagen bundles and scored from one to three. The presence of senescence was defined by P21 or P16 positivity without co-localised Ki-67. The expression of P16, P21, and CTGF on fibroblasts and endothelia was scored from zero to two. The semi-quantitative score was evaluated independently in the superficial and deep dermis by agreement of two researchers (MRD and YHC). EndMT was assessed by co-localisation of CD31 and α-SMA double staining by immunofluorescence, and enclosure of ERG positive endothelial cell nuclei by α-SMA stained cytoplasm under bright field. Lymph vessels were identified by D2-40 (podoplanin) staining, and density was assessed. Results In total, skin biopsies from 18 SSc patients and four age-matched healthy controls were investigated. The characteristics of the patients are listed in Table 1. The dermal fibrosis score revealed a correlation with modified Rodnan skin score (Figure 1A). Table 1. Clinical features of 18 patients with systemic sclerosis Age, median (IQR) 47 (21) Female, n (%) 11 (61) Biopsy site, n (%) Upper limb 3 (17) Trunk 6 (33) Lower limb 8 (44) Type of SSc, n (%) Limited cutaneous SSc 13 (72) Diffuse cutaneous SSc 5 (28) mRSS, median (IQR) 4 (9) Anti-topoisomerase I antibody positive, n (%) 3 (17) Anti-RNA polymerase 3 positive, n (%) 2 (11) Anti-centromere positive, n (%) 3 (17) SSc, systemic sclerosis; IQR, interquartile range; mRSS, modified Rodnan skin score. Figure 1. (A) The modified Rodnan skin score (mRSS) was higher in skin biopsies with severe fibrosis. (B) While merging superficial and deep dermis semi-quantitative scores, increased senescence marker was associated with increased CTGF expression on fibroblasts. (C) Endothelial to mesenchymal transition (EndMT) was more in systemic sclerosis skin without difference between groups with various fibrosis severity. (D)EndMT was associated with increased senescence markers on fibroblasts. Senescence markers showed higher expression on fibroblasts from highly fibrotic dermis than healthy control. The median (IQR) percentage of P16 positive fibroblasts in dermis was 0.7 (0.5) in healthy controls, 0.9 (0.7) in grade one fibrosis, 0.5 (0.5) in grade two fibrosis, and 6.0 (4.6) in grade three fibrosis (p = 0.144). The median (IQR) percentage of P21 positive fibroblasts in dermis was 3.3 (7.0) in healthy controls, 10.3 (11.8) in grade one fibrosis, 33.2 (19.2) in grade two fibrosis, and 32.9 (40.7) in grade three fibrosis (p = 0.042). Moreover, increase of senescence markers on fibroblasts was associated with increased CTGF expression (Figure 1B). The density of blood and lymphatic vessels did not correlate with fibrosis severity. Still, the frequency of EndMT was significantly higher in patients with SSc, although it did not differ between groups with various fibrosis severity (Figure 1C). EndMT was not observed in lymphatic vessels (i.e. there was no co-localisation of D2-40 and α-SMA). Finally, the frequency of EndMT was increased with stronger fibroblast senescence (Figure 1D). Conclusion EndMT and fibroblast senescence were more prominent in SSc skin compared to healthy control skin. Strong fibroblast senescence was associated with EndMT. Fibroblast senescence might facilitate EndMT and be involved in SSc fibrosis. Disclosure of Interests Yu-Hsiang Chiu: None declared, Julia Spierings Grant/research support from: J. Spierings has received a grant from Boehringer., Jacob M. van Laar Consultant of: J. M. van Laar has received honoraria from Abbvie, Arxx Tx, Galapagos, Gesyntha, Leadiant, and Roche., Grant/research support from: J.M. van Laar has received grants from Boehringer, Astra Zeneca, MSD, and Roche., Jeska de Vries-Bouwstra: None declared, Marijke van Dijk: None declared, Roel Goldschmeding: None declared.